In systems of diverse origin, the long-chain saturated fatty acids are formed from acyl CoA derivatives by the concerted action of acetyl CoA carboxylase (ACC) and fatty acid synthetase (FAS). In the cell-free extracts prepared from different micro-organisms, both ACC and FAS have been reported to be present in the dissociated state or they may individually exist as tightly associated complexes. In our investigations on Propionibacterium shermanii, we find that the cell- free extracts of these bacteria contain FAS in the dissociated State. The FAS of these bacteria has been partially resolved by ammonium sulfate fractionation and requires exogenous addition of acyl carrier protein for the elaboration of fatty acids from acetyl and malonyl CoA in the presence of other requisite components. Thus, this enzyme appears to resemble the FAS system of Escherichia coli. Fractionated cell-free extracts of propionic acid bacteria grown in the presence of C14-biotin contain biotinyl peptide(s) of molecular weight considerably larger than the biotin carboxyl carrier protein of oxaloacetate transcarboxylase. Furthermore, our investigations reveal that the partially resolved extracts of these bacteria contain an avidin-sensitive and extremely labile enzyme which catalyzes an ATP- dependent fixation of CO2 into acetyl CoA. This enzyme can also carboxylate propionyl CoA by a similar mechanism. To our knowledge, this is the first report demonstrating the presence of another biotinyl enzyme in these bacteria in addition to the well-studied biotinyl enzyme, oxaloacetate transcarboxylase.